The long-term objective of this research is to understand how hepatitis B virus (HBV) and its relatives in the family Hepadnaviridae cause disease, specifically chronic hepatitis and hepatocellular carcinoma (HCC). Woodchuck hepatitis virus closely resembles HBV in genetics and pathogenesis, causing both chronic infection and HCC, and is used as an experimental model for the human disease. The hepadnavirus genome codes for four types of polypeptides, three of which are accounted for as components of the virion. The fourth gene, X, has as yet no known function in virus replication, but is able to trans-activate other genes in transfected cells and is, therefore, suspected to be involved in carcinogenesis. In addition the Principal Investigator has recently obtained evidence for a novel spliced messenger RNA produced by WHV. The genetic function of this mRNA is not yet known, but it may be involved in the expression of the X protein or of another as yet unidentified gene product. This project is directed toward defining the functions of both the spliced mRNA and the X gene. The approach will be to generate mutations which destroy or modify the function of the X polypeptide of WHV and of the spliced mRNA, by means of site-specific mutagenesis of cloned WHV DNA. The mutated genomes will be transfected into liver cell lines which are known to support production of infectious virus, and tested for production of structurally complete virus. Mutant virus produced in this way will then be tested for infectivity in vivo by inoculation into adult woodchucks. Mutants that are thus proven to be infective will be inoculated into newborn woodchucks, which will be followed to determine the frequency with which they develop chronic infection and subsequently HCC. This research project will identify viral genetic factors that influence the infectivity and pathogenesis of WHV. This will provide insight into the biochemistry of virus replication, immunology and other host factors, and virus variants that differ in virulence. This new knowledge will be directly applicable to the treatment and prevention of HBV infection in humans.